DESCRIPTION: The goal of this project is to improve our understanding of the molecular mechanisms underlying the exoendocytotic cycle of synaptic vesicles at synapses. More specifically, the project will address the role of phosphoinositide metabolism in the vesicle cycle. A key function of phosphoinositide turnover in receptor mediated signaling is well established. In addition it is now clear that phosphoinositides also mediate regulated interactions between membranes and submembranous scaffolds, thus regulating a variety of cellular processes, including actin dynamics and membrane traffic, including membrane traffic at the synapse. This application plans to further test the hypothesis that the exo-endocytotic cycle of synaptic vesicles correlates with a cycle of phosphoinositide synthesis and hydrolysis. It will test the prediction that PI(4,5)P2 is required for the formation of clathrin coated vesicles and for the polymerization of actin at endocytic zones, while dephosphorylation of this phosphoinositide is needed for an efficient clathrin uncoating reaction, inhibition of actin polymerization and the regeneration of new synaptic vesicles. We plan to further investigate a putative role in uncoating of synaptojanin 1 (a presynaptic phosphoinositide phosphatase), to determine whether its homologue synaptojanin 2B has a similar function, to perform a structure-function characterization of these enzymes and to elucidate the function of synaptojanin interacting partners, primarily of members of the endophilin family. We also plan to investigate where in the vesicle cycle, and by which enzyme(s), PI(4,5)P2 is regenerated after the action of synaptojanin. Finally, we plan to perform a systematic characterization of the phosphoinositides present in living nerve terminals in resting conditions and after manipulations which stimulate or perturb synaptic vesicle traffic. These studies will involve mouse genetics, studies in cultured cells and synaptosomes as well as in cell free systems. Since mechanisms of vesicular traffic are highly conserved, these studies will not only shed new light on mechanisms of synaptic transmission, but also, more generally, on vesicular transport along the secretory and endocytic pathways of all cells.